The determination of serum triglyceride levels is becoming increasingly important in the diagnosis of several types of hyperlipemia and atherosclerotic heart disease (Kahlke, W. Med. Wscht. 91, p. 26 (1966), Kuo, P. T. and Basset, D. R., Amer. Intern. Med., 59, p. 465 (1963). Conventional procedures for serum triglyceride determination involve hydrolyzing the triglyceride to liberate glycerol and treating the glycerol with various reagents to produce a compound that can be quantitated spectrophotometrically. Generally hydrolysis is achieved using a base, however, U.S. Pat. Nos. 3,703,591 to Bucolo et al and 3,759,793 to Stork et al describe enzymatic techniques using a lipase alone ('793) or in combination with a protease ('591) to achieve hydrolysis. Other non-enzymatic saponification techniques are described in German Pat. Nos. 2,229,849 and 2,323,609.
Currently three enzymatic methods are conventionally used for the determination of glycerol from whatever source. These are as follows:
__________________________________________________________________________ (a) Method of Garland and Randle (Garland, P. B. and Randle, P. J. Nature, 196 p. 987-988 (1962)) ##STR1## ##STR2## ##STR3## (b) Weiland's Method (Weiland, O. Biochem Z., 329 p.313 (1957) ##STR4## ##STR5## (c) Glycerol Dehydrogenase Method (Hagen, J. H. and Hagen, P. B. Can. J. Biochem. and Physiology, 40 p. 1129 (1962)) ##STR6## __________________________________________________________________________
Modifications of the method of (a) are also described in German Pat. No. 2,665,556, British Pat. No. 1,322,462 and U.S. Pat. No. 3,759,793. In all cases NADH production or disappearance is measured at 340 nm in a U.V. spectrophotometer. Method (a), utilized in many commercial "kits," is a three enzyme sequence and NADH disappearance is measured. Method (b) involves a two enzyme sequence in which NADH production is measured as is the case with the single enzyme glycerol dehydrogenase reaction (method (c)). The latter two procedures are extremely pH-sensitive and subject to error if strict pH control is not maintained. Also, in all three methods (especially method (a)) stability of not only the diagnostic enzymes but also the cofactor, NADH, is a major concern. Errors in current enzymatic methods are discussed in greater detail in Chen, H. P. and El-Mequid, S. S., Biochemical Medicine, 7, p. 460 (1973).
Another method for triglyceride analysis is described in German Pat. No., 2,139,163. The method of this patent involves saponification of the triglycerides, oxidation of the resulting glycerol to formaldehyde and reaction of the formaldehyde with ammonia and a stable, water- and alcohol-soluble, colorless metal complex of acetylacetone to produce a colored compound.